Our laboratory studies molecular and cellular mechanisms of vision.
Most of our work is centered on the vertebrate photoreceptor cell, a
sensory neuron responsible for the light detection in the eye. At this
time, we are pursuing the following experimental directions.
I. REGULATION OF LIGHT SIGNALING
The basic functions of photoreceptors are to capture photons, to
generate a second messenger signal, to translate this signal into a
change in electrical activity and, finally, to transmit this
information to the secondary neurons in the retina through modulation
of synaptic release. Because the function of this cell is so
well-defined and because it is uniquely suitable for study using modern
multi-disciplinary approaches, the photoreceptor is an almost unmatched
model system for elucidating fundamental issues in molecular
neuroscience and cell signaling. The most mature direction of our
laboratory addresses how each of these steps in information flow, from
photon capture to synaptic release, is regulated on the molecular
level. We are particularly interested in learning how signal
amplification, response duration and light adaptation are performed by
the visual signal transduction cascade illustrated in this figure.
Schematic of the phototransduction cascade activation and deactivation
II. HOW TO BUILD A LIGHT-SENSITIVE ORGANELLE?
Photoreceptors are highly compartmentalized neurons, with all molecular
events responsible for generating light-signals confined to the outer
segment, a ciliary organelle containing a stack of flattened membrane
vesicles (“photoreceptor discs”) enclosed within a plasma membrane.
Building this organelle involves two interconnected processes –
formation of its unique anatomical structure and populating it with a
unique set of signaling and structural proteins. We are studying both
of these processes. Our first goal is to reveal the intracellular
trafficking pathways responsible for specific protein delivery to the
outer segment. Almost everything we know today about outer segment
protein trafficking relates to that of the visual pigment, rhodopsin.
The mechanisms responsible for delivery of other outer segment-resident
proteins remain elusive. It is not even clear whether delivery of other
proteins utilizes the components of rhodopsin trafficking pathway, or
several unique pathways coexist in photoreceptors. These conceptually
distinct options are explored in our ongoing experiments. Our second
goal is to elucidate the complex process of photoreceptor disc
morphogenesis, which starts with evagination of the plasma membrane at
the outer segment base, followed by lateral membrane outgrowth,
flattening, and, in the cases of rods and mammalian cones, subsequent
disc enclosure. We are aiming to identify proteins responsible for
performing each of these specific tasks and to understand how they
function as highly coordinated molecular ensemble.
electron micrograph of the mouse rod outer segment and the connecting
cilium bridging the outer segment with the rest of the cell. The newly
forming discs are “open” and have higher electron density that the
mature, enclosed discs.
A frog rod photoreceptors expressing fluorescent plasma
shown in green. The outer segment is stained in red and nucleus in blue.
III. PHOTORECEPTOR PATHOBIOLOGY
Our studies of photoreceptor cell biology go hand-in-hand with
understanding pathobiological processes underlying degenerative
diseases of the retina, which lead to blindness in animals and human
patients. Currently we pursue two directions. The first explores the
novel concept that a major cellular stress factor contributing to
photoreceptor cell death in multiple forms of retinal degenerative
disorders is proteasomal overload, i.e. insufficient capacity of the
ubiquitin-proteasome system to process abnormally large quantities of
misfolded and/or mistrafficked proteins associated with these
conditions. The second addresses the functional role of PRCD
(Progressive Rod-Cone Degeneration), a small protein whose mutations
serve as a leading cause of blindness in dogs and are also identified
in human patients undergoing progressive visual loss. Our recent
proteomic study identified PRCD as a constitutive component of
photoreceptor discs. Our current experiments aim to pinpoint its
specific role in these membranes and to understand why PRCD mutations
cause photoreceptor degeneration and blindness.
Intracellular accumulation of the fluorescent reporter protein, UbG76V-GFP (green), associated with proteasomal overload in
photoreceptor cells degenerating due to various mutations. Outer segments are highlighted by a red lectin marker.
IV. MASS SPECTROMETRY
One of the most powerful approaches to understanding the function of a
cellular organelle is to know its protein composition. This is
certainly true for photoreceptors, and we are engaged in determining
protein compositions of their subcellular compartments using high-end
mass spectrometry. Our favorite experimental strategy is protein
correlation profiling, a powerful approach to analyze multi-protein
complexes or organelles that can be fractionated but not purified to
homogeneity. We recently published the unique proteome of photoreceptor
discs. Our ongoing and planned projects include identification of
protein compositions of the plasma membrane encapsulating the outer
segment, the connecting cilium bridging the outer segment with the
photoreceptor cell body, and transport vesicles carrying building
materials from biosynthetic membranes to the outer segment.
Eleven transmembrane proteins uniquely residing in photoreceptor disc membranes.